Calcitonin (CT) is the most potent and rapidly acting known inhibitor of bone resorption that targets the osteoclast (OC) directly. CT was the first therapeutic agent used to inhibit excessive bone resorption in osteoporosis and other conditions. It is still used, and new formulations for treating osteoarthritis and osteoporosis are currently in development, despite the fact that a reported loss of efficacy with time has somewhat limited clinical use. Thus, new information about the mechanisms by which CT affects OC function has potential therapeutic importance. In vitro, CT rapidly inhibits OC motility and disrupts the podosome belt, the cytoskeletal attachment feature of mature resorbing OCs, accounting for much of CT's inhibition of bone resorption. During the past funding period we showed that CT inhibits OC spreading, motility and bone resorption in large part by inhibiting the protease calpain, and that inhibiting calpain also promotes the recycling of the CT receptor to the cell surface by filamin. These results are a major breakthrough in understanding how CT regulates OC cytoskeleton organization and podosome function. This renewal application seeks to characterize the calpain-dependent mechanisms of CT action, focusing on how CT regulates calpain activity in OCs and how calpain regulates OC attachment and motility, which will provide novel insights into the regulation of osteoclast function in bone remodeling. The Specific Aims of this proposal are therefore: 1. To further elucidate the mechanisms by which calpain modulates podosome function, cytoskeletal organization, cell adhesion and motility, specifically focusing on proteins (cortactin, talin, Pyk2, filamin) that we or others have identified as calpain substrates that play key functional roles in adhesion complexes in osteoclasts; 2. To further characterize the coupling of the CTR to calpain in OCs and the calpain- independent effects of CTR-induced signaling effectors on the function of podosome components; 3. To further characterize the molecular basis of the CTR-filamin interaction, and determine how that interaction affects CTR function, and more specifically, CT's regulation of OC attachment and motility. These studies will reveal previously unappreciated features of CT's mechanisms of inhibiting bone resorption, and thereby possibly increase CT's therapeutic potential by creating the possibility of rapidly and potently inhibiting bone resorption while avoiding the loss of response.